Electrophoresis is an electrochemical process in which molecules with a net charge migrate in a solution under the influence of an electric current. Traditionally, slab gel electrophoresis has been a widely used tool in the analysis-of genetic materials. See, for example, G. L. Trainor, Anal. Chem., 62, 418-426 (1990). Recently, capillary electrophoresis (CE) has emerged as a powerful separations technique, with applicability toward a wide range of molecules from simple atomic ions to large DNA fragments. In particular, capillary gel electrophoresis (CGE) has become an attractive alternative to slab gel electrophoresis (SGE) for biomolecule analysis, including DNA sequencing. See, for example, Y. Baba etal., Trends in Anal. Chem., 11, 280-287 (1992). This is generally because the small size of the capillary greatly reduces Joule heating associated with the applied electrical potential. Furthermore, CGE produces faster and better resolution than slab gels. Because of the sub-nanoliter size of the samples involved, however, a challenging problem in applying this technology is the detecting and monitoring of the analytes after the separation.
Currently, sophisticated experiments in chemistry and biology, particularly molecular biology, involve evaluating large numbers of samples. For example, DNA sequencing of genes are time consuming and labor intensive. In the mapping of the human genome, a researcher must be able to process a large number of samples on a daily basis. If many capillaries of CE can be conducted and monitored simultaneously, i.e., multiplexed, cost and labor for such projects can be significantly reduced. Attempts have been made to sequence DNA in slab gels with multiple lanes to achieve multiplexing. However, slab gels are not readily amenable to a high degree of multiplexing and automation. Difficulties exist in preparing uniform gels over a large area, maintaining reproducibility over different gels, and loading sample wells. Furthermore, difficulties arise as a result of the large physical size of the medium, the requirement of uniform cooling, large amounts of media, buffer, and samples, and long run times for extended reading of nucleotides. Unless gel electrophoresis can be highly multiplexed and run in parallel, the advantages of capillary electrophoresis cannot produce substantial gain in shortening the time needed for sequencing the human genome.
Significantly, the capillary format is in fact well suited for multiplexing. The substantial reduction of Joule heating per lane makes the overall cooling and electrical requirement more manageable. The cost of materials per lane is reduced because of the smaller sample sizes. The reduced band dimensions are ideal for excitation by laser beams and for imaging onto solid-state array detectors. The use of electromigration injection, i.e., applying the sample to the. capillary by an electrical field, provides reproducible sample introduction with little band spreading and with little labor.
Among the techniques used for detecting target species in capillary electrophoresis, laser-excited fluorescence detection so far has provided the lowest detection limits. Therefore, fluorescence detection has been used for the analysis of chemicals, especially macromolecules in capillary electrophoresis. For example, Zare et al. (U.S. Pat. No. 4,675,300) discussed a fluoroassay method for the detection of macromolecules such as genetic materials and proteins in capillary electrophoresis. Yeung et al. (U.S. Pat. No. 5,006,210) presented a system for capillary zone electrophoresis with laser-induced indirect fluorescence detection of macromolecules, including proteins, amino acids, and genetic materials.
Systems such as these generally involve only one capillary. There have been attempts to implement the analysis of more than one capillary simultaneously in the electrophoresis system, but the number of capillaries has been quite small. For example, S. Takahashi et al., Proceedings of Capillary Electrophoresis Symposium, December, 1992, referred to a multi-capillary electrophoresis system in which DNA fragment samples were analyzed by laser irradiation causing fluorescence. This method, however, relies on a relatively poor focus (large focal spot) to allow coupling to only a few capillaries. Thus, this method could not be applied to a large number of capillaries, such as 1000 capillaries, for example. This method also results in relatively low intensity and thus poor sensitivity because of the large beam. Furthermore, detection in one capillary can be influenced by light absorption in the adjacent capillaries, thus affecting accuracy.
Attempts have been made to perform parallel sequencing runs in a set of up to 24 capillaries by providing laser-excited fluorometric detection and coupling a confocal illumination geometry to a single laser beam and a single photomultiplier tube. See, for example, X. C. Huang et al., Anal. Chem., 64, 967-972 (1992), and Anal. Chem., 64, 2149-2154 (1992). However, observation is done one capillary at a time and the capillary bundle is translated across the excitation/detection region at 20 mm/sec by a mechanical stage.
There are features inherent in the confocal excitation scheme that limit its use for very large numbers (thousands) of capillaries. Because data acquisition is sequential and not truly parallel, the ultimate sequencing speed is generally determined by the observation time needed per DNA band for an adequate signal-to-noise ratio. Having more capillaries in the array or being able to translate the array across the detection region faster will not generally increase the overall sequencing speed. To achieve the same signal-to-noise ratio, if the state-of-the-art sequencing speed of 1000 nucleotides/hour/lane is used, the number of parallel capillaries will have to be reduced proportionately regardless of the scan speed. Moreover, the use of a translational stage can become problematic for a large capillary array. Because of the need for translational movement, the amount of cycling and therefore bending of the capillaries naturally increases with the number in the array. It has been shown that bending of the capillaries can result in loss in the separation efficiency. This is attributed to distortions in the gel and multipath effects. Sensitive laser-excited fluorescence detection also requires careful alignment both in excitation and in light collection to provide for efficient coupling with the small inside diameter (i.d.) tubing and discrimination of stray light. The translational movement of the capillaries thus has to maintain stability to the order of the confocal parameter (around 25 .mu.m) or else the cylindrical capillary walls will distort the spatially selected image due to misalignment of the capillaries in relation to the light source and photodetector.
Further, the standard geometry for excitation perpendicular to the axis of the capillary requires the creation of an optically clear region in the capillary. This makes the capillary fragile and complicates the preparation of capillaries for use. Moreover, the excitation path length, and hence the fluorescence signal, is restricted to the small diameter of the capillary. Therefore, there is a need for a fluorescence detection system that can be utilized to analyze a large number of samples substantially simultaneously without bulky equipment and unduly complicated procedures such as careful alignment.